Intracellular proteases of the AAA+ and HtrA families remove toxic proteins from cells and participate in cellular processes that promote health and prevent disease. Because protein synthesis is energetically expensive and proteolysis is irreversible, protein degradation must be carefully regulated to conserve precious cellular resources. Growing evidence suggests that some AAA+ and HtrA proteases can also help proteins refold and/or prevent their aggregation, but how a single enzyme can carry out these activities in addition to the seemingly antagonistic activity of protein degradation is unknown and represents the major goal of this proposal. Building on our experience and results from the previous funding period, we will use a combination of structural biology, biochemistry, protein-engineering, and molecular genetics to determine how protease versus chaperone activities are mediated by changes in protein conformation or assembly and are regulated by the binding of substrates and adaptor proteins. For example, we will determine the regulatory events and features that allow the archaeal AAA+ Cdc48 enzyme to function either as a disassembly chaperone or as a part of the Cdc48?20S proteasome. We will also test if this alternative proteasome operates in eukaryotic cells. Notably, inhibitors of the eukaryotic 20S peptidase are used to treat multiple myeloma and are active in animal models of autoimmune and inflammatory disease, and knowledge of which proteasomes are the actual targets would be an important advance. Our studies will identify mutations that eliminate just chaperone or just protease activity, allowing the importance of each activity to be quantified, identify new substrates and adaptors, reveal connections between enzyme function and biological mechanism, and allow the rational design of highly selective inhibitors for use in basic science and medicine. In pathogenic bacteria, enzymes of the AAA+ and HtrA families are required for virulent infection and are potential antibiotic targets. Changes in expression levels or malfunction of AAA+ and HtrA enzymes in humans have been linked to arthritis, cancer, vascular disease, macular degeneration, myopathy, diabetes, dementia, retardation, Parkinson's disease, and Alzheimer's disease. Although the molecular mechanisms are largely unknown, proteases and chaperones of the AAA+ and HtrA families have also been shown to play critical roles in a large number of diverse cellular pathways, including antigen presentation, cell-cycle progression, DNA repair/recombination, Golgi and nuclear membrane reassembly, autophagy, mitochondrial function, apoptosis, and sensing and combating stress, mutation, infection, and aging. Our studies will establish or clarify mechanistic connections between AAA+ and HtrA enzymes and biological function and dysfunction.